Efficient DNA extraction and sequencing protocol for keratinised materials of animals

Keratinised tissues represent a valuable non-invasive biological source of mitochondrial and nuclear DNA and hold significant potential for genomic research. However, studies using genomic DNA from keratinised tissues (kmDNA) remain considerably limited, particularly in non-human species, with most studies focusing on human hair. The limited understanding of kmDNA quality constrains its application, particularly in contexts where high-quality DNA is scarce, such as in wildlife conservation and historical research. Thus, this study aimed to develop an efficient DNA extraction and whole-genome sequencing protocol for isolating DNA from diverse keratinised materials. The protocol incorporates several key improvements that enhance the recovery of endogenous DNA from highly degraded samples. This optimised workflow allowed the recovery of DNA fragments as short as 20-30 bp and increased the proportion of endogenous DNA (R_m) by over 20 % on average. The refined library preparation strategy increased sequencing chip utilisation (the ratio of actual output data to its theoretical maximum output) by more than 50 % in some samples. This study provides an optimised sequencing protocol for the systematic evaluation of DNA quality across common keratinised materials, establishing a robust methodological framework for the genetic analysis of complex degraded samples (e.g. historical or field-collected samples). These advancements significantly expand the practical boundaries of molecular biological research under non-ideal conditions.